Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes

This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020). [Display omitted] •FLAG-tagged proteins and their interactome can be immunopurified using FLAG antibodies•FLAG peptide elution enriches protein complexes and prevents elution of immunoglobulins•This protocol validated for ribonucleoprotein enrichment has broad application•Mass spectrometry and bioinformatics can reveal functions of a protein of interest This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest.